Mammalian cell culture has emerged to become an indispensable technology for the production of a number of the desirable end products. Non-limiting examples of such desirable end products include human therapeutic biologicals and/or biomass materials, which are made in large-scale animal cell cultures grown in bioreactors. In some cases, the demand for these biologicals and/or biomass materials is on the order of tens to hundreds of kilograms per year, making mammalian cell culture on the scale of 10,000 liters, or higher, routine.
The bioreactors are designed especially for mammalian cell culture and for their ability to support cell growth and end product production under batch culture conditions. The industry has experienced challenges, however, in achieving maximum viable cell densities and desired end-product production. Differences in vessel and agitator geometry and aeration modes of operation have not completely solved this problem.
It is believed that one cause of such limitations in the scaled-up processes is due, at least in part, to the loss of cells due to lethal, mechanical damage in the growth vessels. This lethal damage is generally believed to be the result of hydrodynamic and interfacial forces acting on the cells and these stresses influence both the viability and specific production rate of the cells and thus the overall productivity of the reactors.
In certain conditions, this cell death is caused by the attachment of cells to gas-liquid interfaces at the top surfaces of the vessels where the aqueous media, gas bubbles (typically introduced in the bottom of the vessel and subsequently rise to the top), and air and/or gas above the aqueous media come into contact. Hydrodynamic and interfacially driven forces are very high at these interfaces, especially when a bubble ruptures, but is also high in the foam layer where bubbles drain and coalesce.
It has been experimentally reported that under certain conditions, the presence of cells attached to bubbles is a major cause of cell death in sparged cell cultures. The cells that die, for example, during the bubble burst can come from three sources: cells suspended near the bubble; cells trapped in the bubble lamella; and cells that attached to the rising bubble. This cell attachment can depend on cell radius, bubble radius, and cell-bubble attachment time.
In addition, there are concerns with respect to potential cell damage as a result of gas sparging and foam formation that can occur in large scale-up productions. This is especially true as cell concentrations during such scale-ups can increase to 10 million cells per milliliter and higher in fed-batch and perfusion cultures.
Another concern is the interactions among cells, bubbles that form and disperse within the cell cultures. This becomes of increasing importance, as there is a greater demand for the safe, effective and efficient design and operation of large-scale production of biologicals and/or biomass materials from cell cultures. Another concern is how to develop protective additives that can be useful over a wide range of additive conditions and cell concentrations.
Many researchers have observed sparging-related cell damage. These reports all underscore the concept that cell-bubble interactions play a much more important role in physical cell damage than pure agitation.
In particular, the high demand for some of these end products, such as antibodies, has inevitably lead to the requirement of more efficient manufacture processes, particularly high productivity and high cell density. However, mammalian cells are sensitive to the surrounding environment, including the concentration of ammonia, lactate, dissolved carbon dioxide osmolality, and pH. Nevertheless, through careful and systematic development, industrial fed-batch or perfusion cell culture processes have been able to increase product titers, from typically around 50 mg/L to over 5 g/L over a two decade period.
Along with a better understanding of cell metabolism from a biological perspective, significant efforts have been made to explore physical challenges in mammalian cell culture, including mixing, mass transfer, and cellular sensitivity to hydrodynamic forces. While a number of systems have been proposed for large scale culture, the stirred tank bioreactor with gas sparging is the commercial system of choice. Such a system, while simpler to implement and operate than other more complex systems, creates gas-liquid-solid (cell) multiphase environments within the vessel.
The presence of surface-active compounds, including surfactants and proteins, makes these interfaces highly complex; however, these interfaces are fundamental to the gas-liquid mass transfer (O2 supplement and CO2 removal). Unfortunately, these gas-liquid-cell interfaces (cell-gas adhesion, foam formation, cells trapping with in the foam, and protein/lipid adhesion to gas-liquid-solid interfaces) also occur when sparging is used. Unfortunately, the current understanding of interfacial phenomena in cell culture processes is predominately based on empirical studies. In particular, a number of additives have been examined for their protective effect on bubble-associated cell damage. These additives include, for example, fetal bovine serum (Kunas and Papoutasakis, 1989), the Pluronic® series of surfactants (Murhammer and Goochee, 1990); methyl cellulose (Goldblum et al., 1990), Dextran (van der Pol et al., 1995), polyethylene glycol (Michaels and Papoutsakis, 1991) and polyvinyl alcohol (Michaels et al., 1992). Among them, the Pluronic F-68® (PF-68®) surfactant, first advocated in the 1960's, is still the most commonly used additive (Swim and Parker, 1960; Runyan and Geyer, 1963; Kilburn and Webb, 1968). There is no doubt that the PF-68® surfactant contributes significantly (potentially vital) to the success of industrial mammalian cell culture in bioreactors.
The PF-68™ material is a nonionic surfactant with triblock structure consisting of hydrophobic polypropylene oxide) center and two hydrophilic poly(ethylene oxide) tails, and which does not have a distinct critical micelle concentration (CMC). It has an average molecular weight of 8400. Even though a number of protective mechanisms of the Pluronic PF-68® surfactant have been proposed (Chisti, 2000; Wu, 1995), the ability of the PF-68® material to inhibit cell-bubble attachment is considered the primary mechanism. Chattopadhyay et al. (1995b), suggested that this inhibition of cell-interface adhesion is the result of the PF-68® material significantly decreasing the surface tension of the gas-liquid interface such that adhesion to the gas-liquid interface is thermodynamically unfavorable. The interaction and structure of block, non-ionic copolymers (such as PF-68®) with air-water interfaces is a subset of a significant area of research in the general areas of surfactants and air/water interface interactions.
With respect to cells, Ma et al. (2004) recently quantitatively studied the interactions among cells, bubbles, and PF-68® over a broad range of PF-68® and PER.C6® cell concentrations. The cell concentration in the foam liquid decreased dramatically with the increase of PF-68™ concentration; however, as the cell concentration increased (on the order of 107 cells/ml, or higher) even at high PF-68 concentration of greater than 1 g/L, over 1000 PER.C6® per bubble became trapped in the foam layer.
As the final cell concentration of commercial mammalian cell culture systems continue to increase, it is apparent that the effectiveness of the PF-68® surfactant will diminish. While it can be argued that one can tolerate some loss of productivity of a relatively small number of cells in the foam, even the lysis of this small number, with the subsequent release of the intracellular components into the media is an unwelcomed occurrence. In addition, it is highly desirable to have other, effective, alternatives to the PF-68® surfactant.
In addition to their intended function at gas-liquid interfaces, surfactant additives can have a significant effect on the cell membrane. The mammalian cell membrane is a presumably thermodynamically-controlled and self-assembled bilayer consisting of phospholipids, triglycerides, cholesterol, and trans-membrane proteins. The membrane structure is dynamic with the lateral diffusion and flip-flop of lipid components occurring. It is generally believed that specific surfactants can efficiently compete with the interactions between membrane lipids and proteins, ranging from incorporation and/or partitioning of the surfactant into the cell membrane, to the outright dissolving of the membrane structure (Neimert-Andersson et al. 2006).
Therefore, one of the most important criteria with respect to a material that will prevent negative cell gas adhesion is that such material should not be harmful to mammalian cells. At least two mechanisms have been proposed to explain the most harmful of surfactant interactions beyond direct plasma membrane solubilization: flip-flop and micellar attack (Maire et al., 2000). In the former, non-micellar surfactants penetrate the membrane and cause damage by disrupting the cell membrane structure, including flipping lipids inside out. In the latter, membrane compounds are transferred directly from the outer side of membrane bilayer into surfactant micelles. In spite of the importance of surfactants to protect, and alternatively, solubilize mammalian cells, the selection of surfactants, both with respect to type and concentration selection and optimization is still a semi-empirical process.
In yet another area or research, despite a fundamental understanding of the interactions of surfactants with cells, and specific molecules of interest (i.e., hydrophobic nutrients and drugs), significant interest exists in the drug delivery research community to further develop surfactants that solubilize drugs with low water solubility, yet do not cause any cellular damage. Historically, polyoxyethylene-based surfactants have been one type of surfactant used for this purpose; however, there are number of disadvantages. Consequently, a number of studies exist investigating, and suggesting, other surfactants for drug delivery. Thus, while there is a different purpose than the prevention of cell adhesion to gas medium interfaces, many of the attributes required for an optimum drug delivery surfactant are similar to prevention of cell adhesion surfactant.
The discussion of various publications cited herein and other prior knowledge does not constitute an admission that such material was published, known, or part of the common general knowledge.